PROJECT SUMMARY/ABSTRACT Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of patients with leukemia, yet robust responses to this modality in solid tumors have been lacking. As a result, most patients with relapsed or refractory solid tumors remain in dire need of alternative therapy. Given the inhospitable solid tumor microenvironment, we are unlikely to achieve robust responses without the most highly potent CAR T cells. My previous studies have demonstrated that CAR T cell potency can be enhanced by increasing the affinity of CAR for its cognate antigen. Further investigation revealed that in particular cytotoxicity was enhanced, whereas T cell survival and central memory (Tcm) differentiation (both important in CAR T clinical persistence and efficacy) were actually impaired in the high affinity CAR T cells. The inverse influence of antigen binding affinity on cytotoxicity and persistence raises questions as to the cellular signaling events underlying these discordant effects. The findings also highlight the possibility that by reversing these decrements in survival and memory in the high affinity cells, CAR T cell potency could be unleashed even further. The central hypothesis to be tested here is that increased CAR affinity results in faster and more sustained activation of downstream T cell signaling pathways, enhancing cytotoxic granule release yet overly engaging signaling molecules such as ERK and Akt, tipping the cellular balance away from survival and memory differentiation and toward cell death. To test the hypothesis, first I will use selected panels of affinity variants to evaluate the relationship between CAR affinity and the activation kinetics of key signaling pathways by quantitative Western blot and phospho-protein flow cytometry. Then using the same panels of affinity variants, I will evaluate the impact of CAR affinity on both cytotoxic granule release using microscopy as well as T cell survival and memory differentiation in vitro using flow cytometry. Lastly, I will identify and evaluate targetable mediators of the survival balance operating in high affinity CAR T cells in vitro and in vivo. To do so, I will compare the affinity variant CAR T cells? survival and Tcm differentiation in vitro and in vivo in the presence or absence of specific inhibitors of pathways (MEK, Akt, and Fas) that when over-activated can impair T cell survival or memory differentiation. These studies are expected to yield highly potent CAR T cells equipped to address the challenge of solid tumors where both efficient killing and robust persistence are necessary for efficacy. The training plan developed for this proposal is designed to provide me with skills such as advanced microscopy techniques that are necessary to best address the questions raised. Also incorporated into the training plan is mentorship from experts in the CAR field, an advisory committee with special expertise in lymphocyte signaling and imaging, formal didactic experiences in career development, and an overall training environment that will help me to develop into an independent physician- scientist.